Difluorophosphate as a 19F NMR probe of erythrocyte membrane potential

Erythrocyte membrane potential can be estimated by measuring the transmembrane concentration (activity) distribution of a membrane-permeable ion. We present here the study of difluorophosphate (DFP) as a ¹⁹F NMR probe of membrane potential. This bicarbonate and phosphate analogue has a pK_a of 3.7±0.2 (SD, n = 4) and therefore exists almost entirely as a monovalent anion at physiological pH. When it is incorporated into red cell suspensions, it gives two well resolved resonances that arise from the intra- and extracellular populations; the intracellular resonance is shifted 130 Hz to higher frequency from that of the extracellular resonance. Hence the transmembrane distribution of DFP is readily assessed from a single ¹⁹F NMR spectrum and the membrane potential can be calculated using the Nernst equation. The membrane potential was independent of, DFP concentration in the range 4 to 59 mM, and haematocrit of the cell suspensions of 31.0 to 61.4%. The membrane potential determined by using DFP was 0.94±0.26 of that estimated from the transmembrane pH difference. The distribution ratios of intracellular/extracellular DFP were similar to those of the membrane potential probes, hypophosphite and trifluoroacetate. DFP was found to be transported across the membranes predominantly via the electrically-silent pathway mediated by capnophorin. Using magnetization transfer techniques, the membrane influx permeability-coefficient of cells suspended in physiological medium was determined to be 7.2±2.5 × 10^–6 cm s^–1 (SD, n=4). Offprint requests to: P. W Kuchel